Rapid and Efficient Purification of Functional Collectin-12 and Its Opsonic Activity against Fungal Pathogens.

Collectin-12 (collectin placenta 1, CL-P1, or CL-12) is a newly identified pattern recognition molecule of the innate immune system. Recent evidences show that CL-12 plays important roles not only in innate immune protection against certain clinically important pathogens but also in scavenging of host molecules, leukocyte recruitment, and cancer metastasis. Furthermore, CL-12 has been shown to be associated with the pathogenesis of human diseases such as Alzheimer's disease and multiple sclerosis lesion development. Therefore, the functional consequence of CL-12 remains intriguing and awaits further elucidation. However, available protocols for the purification of recombinant CL-12 with high purity are laborious and inefficient and hamper further functional studies. Here, we report a simple, rapid, and efficient solution to obtain biologically active CL-12 with high purity. We established stable transfected Flp-In™-CHO cells expressing the recombinant CL-12 extracellular domain in high amounts. Recombinant CL-12 was purified from cell culture supernatants using a 3-step rapid purification procedure utilizing disposable affinity and ion exchange minicolumns. Purified recombinant CL-12 adopted an oligomeric structure with monomers, dimers, and trimers and retained its binding capacity towards the A. fumigatus strain that has been described before. Furthermore, we demonstrated the opsonic properties towards eight clinical isolates of A. fumigatus strains and diverse clinically important fungal pathogens. Purified recombinant CL-12 revealed a differential binding capacity towards selected fungal pathogens in vitro. In conclusion, we demonstrate a rapid and efficient purification solution for further biochemical and functional characterization of CL-12 and reveal opsonic properties of CL-12 towards diverse fungal pathogens.

Heteromeric complexes of native collectin kidney 1 and collectin liver 1 are found in the circulation with MASPs and activate the complement system.

The complement system is an important part of the innate immune system. The complement cascade may be initiated downstream of the lectin activation pathway upon binding of mannan-binding lectin, ficolins, or collectin kidney 1 (CL-K1, alias CL-11) to suitable microbial patterns consisting of carbohydrates or acetylated molecules. During purification and characterization of native CL-K1 from plasma, we observed that collectin liver 1 (CL-L1) was copurified. Based on deglycosylation and nonreduced/reduced two-dimensional SDS-PAGE, we detected CL-K1 and CL-L1 in disulfide bridge-stabilized complexes. Heteromeric complex formation in plasma was further shown by ELISA and transient coexpression. Judging from the migration pattern on two-dimensional SDS-PAGE, the majority of plasma CL-K1 was found in complex with CL-L1. The ratio of this complex was in favor of CL-K1, suggesting that a heteromeric subunit is composed of one CL-L1 and two CL-K1 polypeptide chains. We found that the complex bound to mannan-binding lectin-associated serine proteases (MASPs) with affinities in the nM range in vitro and was associated with both MASP-1/-3 and MASP-2 in plasma. Upon binding to mannan or DNA in the presence of MASP-2, the CL-L1-CL-K1 complex mediated deposition of C4b. In favor of large oligomers, the activity of the complex was partly determined by the oligomeric size, which may be influenced by an alternatively spliced variant of CL-K1. The activity of the native heteromeric complexes was superior to that of recombinant CL-K1. We conclude that CL-K1 exists in circulation in the form of heteromeric complexes with CL-L1 that interact with MASPs and can mediate complement activation.

Purification and electrophoretic characterization of bovine conglutinin.

In this study a two-stage procedure for purification of conglutinin using affinity and ion-exchange chromatography was developed. To isolate conglutinin from bovine serum, its unique ability to bind to complement component iC3b was exploited. Incubation of bovine serum with chromatographic beads (TSK, Toyopearl HW-75 F) at 37 °C allows for iC3b deposition and subsequent binding of conglutinin. A single protein fraction eluted with ethylenediaminetetraacetic acid (EDTA) was then separated on an ion-exchange column in an NaCl gradient. The purification was evaluated by SDS-PAGE and western blotting. Conglutinin analyzed by SDS-PAGE under reducing conditions showed two main bands at 41 and 47 kDa and eight weaker bands. Nonreduced conglutinin appeared as a ladder pattern composed of many fractions ranging from 34 to 630 kDa. The bands at 34, 153, 174, 247, 338 and 387 kDa displayed the highest optical density. In the native conglutinin profile four fractions were observed, and the pI of this protein was below 8.5. The presence of sugar residues in the conglutinin molecule was detected using Schiff's reagent.

A simple two-step purification procedure for the iC3b binding collectin conglutinin.

Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37°C to allow complement activation and iC3b deposition on the beads and subsequent binding of conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81 µg of conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified conglutinin had a high affinity for mannan (K(d)=2.3-3.2 nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.

Isolation and expression of a novel MBL-like collectin cDNA enhanced by LPS injection in the body wall of the ascidian Ciona intestinalis.

Collectins are a family of calcium-dependent lectins that are characterized by their collagen-like domains. Considerable interest has been focused on this class of proteins because of their ability to interact with components of the complement system activating a cascade of events responsible for the activation of the innate immune system. A differential screening between LPS-challenged and naïve Ciona intestinalis has been performed allowing the isolation of a full length cDNA encoding for a 221 AA protein. In silico analysis has shown that this polypeptide displays protein domains with similarities to mannose-binding lectins. A phylogenetic analysis suggested that C. intestinalis MBL has evolved early as a prototype of vertebrate MBL. Real-time PCR assay demonstrated that this gene is strongly activated after LPS injection in the tunica. In situ hybridization performed in LPS-induced animals has shown that this gene is expressed in granular amoebocytes and large granules hemocytes in the inflamed body wall tissue. Finally, an antimicrobial activity of the C. intestinalis MBL has been demonstrated.

Identification and characterization of a novel human collectin CL-K1.

Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca(2+)-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.

A second form of collagenous lectin from the tunicate, Styela plicata.

This study characterised a 90 kDa lectin from an invertebrate chordate, the tunicate Styela plicata. One- and two-dimensional electrophoresis showed that the apparent molecular weight of this protein is maintained under both reducing and non-reducing conditions, suggesting that its native form is a monomer. The 90 kDa lectin was localised within a single type of hemocyte (morula cells), but was secreted from those cells when tunicates were challenged with the inflammatory elicitor, zymosan. Functional studies showed that the 90 kDa protein binds to galactose-based sugars in a divalent cation-dependent manner. Amino acid composition analysis and N-terminal amino acid sequencing indicated that the 90 kDa lectin is related to a previously characterised, collagenous lectin from S. plicata, splic43. However, peptide mass fingerprinting identified numerous differences between the two proteins. This suggests that the 90 kDa molecule represents a novel protein that is involved in host defence.

CL-46, a novel collectin highly expressed in bovine thymus and liver.

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via alpha-helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the alpha-helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans approximately 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development.